Keratinization is the orderly process in which viable epidermal cells (keratinocytes) manufacture soluble precursor proteins and transform them into highly organized, insoluble protein aggregates (keratin) which are retained within epidermal cells as they mature into nonviable, anucleate cells at the skin surface. A similar process is involved in the production of hair keratin. The extreme insolubility of keratins has not been adequately explained, but specific covalent epsilon-(gamma-glutamyl) lysine crosslinking bonds have recently been found in hair keratin (Biochem Biophys Acta 207:342, 1970) and may contribute significantly to its insolubility. These bonds are enzymatically formed by the action of hair follicle transglutaminase. A similar transglutaminase has been highly purified and characterized from nonhair-bearing bovine epidermis (Buxman and Wuepper, submitted for publication) and localized histochemically and immunochemically to upper Malpighian and granular layers of bovine, rat, and human epidermis (Buxman and Wuepper, JID 65:107-112, 1975). The function of this enzyme in epidermis has not been elucidated. We have identified a single preferential epidermal substrate for epidermal transglutaminase in soluble and highly insoluble form, by its ability to incorporate the fluorescent substrate dansyl cadaverine (Clin Res 24:262A, 1976). We now propose to further study and characterize this substrate by chromatographic and electrophoretic methods and to analyze the kinetics of crosslinking of the protein to itself or to other epidermal proteins. Mechanisms for control of substrate synthesis and crosslinking will be investigated, using inhibitors of the enzyme and/or substrate, in vitro and in tissue culture. The substrate will be localized histochemically in tissue by light and electron microscopy. Finally, using human tissue, normal and abnormal substrate and enzyme function will be examined, using biochemical and immunochemical tools developed in animal models.